S277: HYPOFERREMIA CAUSED BY FERROPORTIN DOWNREGULATION IS REGULATED BY NFКB THROUGH HDAC-DEPENDENT MECHANISMS
Authors: O. Marques, N. Horvat, M. C. Lai, L. Zechner, S. Colucci, R. Sparla, S. Zimmermann, C. J. Neufeldt, S. Altamura, J. Zaugg, K. Mudder, J. D. Knopf, M. K. Lemberg, M. Pasparakis, M. W. Hentze, M. U. Muckenthaler
Journal: HemaSphere (2022)

Background: Anemia of Inflammation (AI) is a highly prevalent comorbidity in patients with chronic inflammatory disorders. The underlying pathophysiology is partly explained by hypoferremia that results from iron sequestration in macrophages and the reduction of dietary iron uptake due to decreased iron export mediated by Ferroportin (Fpn). In response to inflammation, Fpn levels are decreased by hepcidin-mediated post-translational and/or transcriptional mechanisms, depending on the type of inflammatory pathway activated. Despite the fact that Fpn transcriptional repression is frequently observed in inflammatory conditions, the regulatory mechanisms involved remained unidentified. Aims: To gain mechanistic insight how Fpn mRNA levels are regulated in response to inflammation. Methods: We conducted a pharmacological and RNAi screen, targeting mediators of TLR2/6 signalling, in bone marrow-derived macrophages (BMDMs) stimulated with the TLR2/6-specific ligand FSL1. Changes in chromatin accessibility were evaluated by chromatin-accessibility Real-Time Polymerase Chain Reaction (CHART-PCR) and Assay for Transposase-Accessible Chromatin using sequencing (ATACseq). Data obtained in cell-based assays were validated in mice injected with FSL1. Results: The screen in BMDMs together with validation experiments demonstrated that the FSL1 induced TLR2 response represses Fpn mRNA levels via MYD88 and TRAF6 signaling transducers. While FSL1 can activate independent JNK, p38 and NFκB signaling branches, we show that Fpn transcriptional repression is specifically dependent on alternative NFκB signaling involving the transcription factor RELB. This contrasts our findings for TLR4 stimulation by LPS, where reduced Fpn mRNA expression depends on classical NFκB signaling, involving the NFкB subunits RELA and p50. Using ATACseq we identified Fpn genomic regions that are altered by inflammatory stimuli. These data show chromatin condensation around the Fpn transcription start site (TSS) upon FSL1 or LPS treatment. Since chromatin condensation is mainly promoted by histone deacetylases (HDACs), we next tested whether Fpn mRNA reduction upon inflammatory cues could be counteracted by treatment with HDAC inhibitors (HDACi). We demonstrate that pre-treatment with the pan-HDACi SAHA (Suberoylanilide Hydroxamic Acid) prevented chromatin remodelling at the Fpn locus and Fpn transcriptional repression in response to inflammation and RELB overexpression, suggesting that the effect of HDACs on Fpn responses occurs downstream of NFкB. In line with these data, NFκB pharmacological inhibition also prevented Fpn chromatin remodeling in the vicinity of the TSS. The findings obtained in BMDMs were extended in mice treated with SAHA prior to FSL1 injection, showing reduced hypoferremia and attenuation of splenic Fpn mRNA repression, while serum hepcidin levels remained unaltered. Summary/Conclusion: These results identify NFκB as an important repressor of Fpn transcription in response to inflammatory signals involving chromatin remodeling in a genomic region near Fpn’s TSS. Importantly, HDAC inhibition attenuates Fpn mRNA repression as well as the resulting hypoferremia in mice. Our findings generate molecular insight about how hypoferremia is generated and demonstrate that Fpn, like hepcidin, is controlled by HDACs.